title: Preparation and characterization of PEGylated amylin.
abstract: Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys(1). The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.
authors.affiliation: School of Pharmacy, Federal University of Rio de Janeiro (UFRJ, CCS), Bss34, Ilha do Fundão, 21941-590, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Luiz Henrique
authors.initials: LH
authors.lastname: Guerreiro
authors.forename: Mariana F A N
authors.initials: MF
authors.lastname: Guterres
authors.forename: Bruno
authors.initials: B
authors.lastname: Melo-Ferreira
authors.forename: Luiza C S
authors.initials: LC
authors.lastname: Erthal
authors.forename: Marcela da Silva
authors.initials: Mda S
authors.lastname: Rosa
authors.forename: Daniela
authors.initials: D
authors.lastname: Lourenço
authors.forename: Priscilla
authors.initials: P
authors.lastname: Tinoco
authors.forename: Luís Maurício T R
authors.initials: LM
authors.lastname: Lima
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D058228:Islet Amyloid Polypeptide; D011092:Polyethylene Glycols; D014357:Trypsin